My name is Ananya Kachru, and I am a rising junior at Amity High School in CT. I decided to participate in the Biological Research program at the Boston Leadership Institute because I am passionate about science research, and because I love biology! My love for biology started when I took a biology class my freshman year at AHS. It quickly became my absolute favorite- I loved it all. Biology intrigues me because it literally is life. It is the science behind every single living thing… it truly amazes me. I am looking forward to taking AP Biology next year! Moreover, ever since the 8th grade, I have been involved with science research. In middle school, I worked on social robotics and created a writing robot called WriteBot (I know, I should have come up with a more creative name!) In my freshman and sophomore years, I worked a lot on behavioral research with cognitive neuroscience. For my junior year, I am looking to expand my horizons and finally do biological research! Science research is truly the crux of modern society, and I believe that the constant revelations being made are truly incredible.
In school, I am involved in several clubs and organizations. I am really, really passionate about debate, and I have been a part of the debate team at my high school since freshman year. I love debate because although it is extremely competitive, it fosters a collaborative environment. Earlier this summer, I was at debate camp. My debate partner and I were fortunate enough to break into quarterfinals at the Calvin Coolidge National Debate Tournament, and all our camp friends helped us intensely prepare for quarterfinals- which I believe really exemplifies the collaborative atmosphere. Also, last year, I founded my school’s Academic Decathlon team along with one of my classmates. Starting the team from ground up and persevering along with 7 amazing teammates to the E-National competition was an incredibly unique experience. ALSO- the science topic for AcDec last year was biology!!! Student Government, Infinite Possibilities (a STEM mentoring program), and Kids v. Cancer are a few of the organizations I am also very involved with at my school. Outside of school, I enjoy volunteering as an assistant dance teacher at my dance school, as a research intern at the Haskins Research Laboratories, and as a teen teacher at a local elementary school (little kids are the best). I also am passionate about youth activism, Kathak dance (Indian classical dancing), and pageantry.
Some fun facts about me:
Favorite food: That is really hard- but probably Margherita pizza from Frank Pepe’s (very famous in CT) coupled with a really good Caesar salad.
Favorite dessert: CHEESECAKE
Favorite TV show: I LOVE Gilmore Girls. But, I have also been a loyal Dance Moms viewer since Season 1. (If any of you are DM fans- I have gone back and forth over Team Chloe and Team Maddie for years… and them both being nominated for TCA Choice Dancer for the second year in a row is so excited ah)!
Favorite movie: The Sisterhood of the Traveling Pants
Favorite color: Hot pink
Favorite Harry Potter house: Gryffindor, of course!
Favorite board game: Either LIFE or RISK
Favorite book: Although I first read it in English Class, I absolutely love The Great Gatsby. I also love the Mother-Daughter Book Club books. I grew up reading them, and they still remain to be some of my favorite books.
Favorite season: Winter! I am a December baby, and literally all of my family’s major occasions happen in the last three months of the year. I also really love New England winters!
Favorite musical: HAMILTON (I am counting down days until I see it!)
Purpose Problem: Appendicitis is the inflammation of the appendix through pus, followed with much pain. It tends to occur when the appendix is blocked- by stool, an infection, foreign bacteria presence, or even cancer. Interestingly, the appendix is often considered to be vestigial and unimportant, but recent studies have shown that there is a particular significance to the appendix. Currently, researchers have found that the appendix essentially acts as a ‘safe house’ for unharmful bacteria. For example, when an individual suffers from dysentery or cholera, the appendix can activate as a ‘reboot mechanism’ for the digestive system. As one in every fifteen people suffer from appendicitis, it is important to design a preventive measure to preserve the appendix.
Importance: It is crucial to solve this problem because as there is currently no functioning preventive measure for appendicitis, so anyone who suffers from the illness must undergo appendectomy or receive antibiotic therapy. Appendectomy is a common procedure, but complications are evidently plausible and it is a significant inconvenience. On the other hand, antibiotic therapy is once again not very effective and causes much pain within individual patients. Providing a preventive measure for the general population will give ease and more comfort. Moreover, determining potential devices in the health field often comes down to cost-effectiveness and quality. If a preventive mechanism is synthetically designed, appendectomy and antibiotic therapy will no longer have to be as prevalent in the health industry and a lot of financial burden will be lifted from potential patients.
Address Problem: The design that I am creating will essentially prevent appendicitis by allowing for a loop between inflammation and de-inflammation. By reengineering Bacteroides, the most abundant bacteria in the appendix, the APD design will largely focus on the prevalence of C-Reactive Proteins (CRPs). These proteins generate in the liver, but are most typically recognized as a marker of inflammation in the cardiovascular system. The APD will utilize the gene that encodes for this protein, in order to express CRPs at the site of inflammation in the appendix rather than the heart. Further, a signal molecule will only bind to a particular receptor for the production of Cluster of Differentiation 47 proteins (CD47) when the CRPs are prevalent in the genetic pathway. CD47 leads to de-inflammation of the appendix. Thereby, whenever inflammation is present, CRPs are produced. When CRPs are produced, CD47 for de-inflammation is synthesized. This design ensures that whenever the appendix is inflamed, it can and will be de-inflamed. With this regular cycle between the two stages, appendicitis can be prevented.
Competing Technologies
Currently, appendicitis is dealt with through two methods- primarily an appendectomy procedure, but recently even an antibiotic therapy (especially with patients enduring uncomplicated acute appendicitis). While both of these methods are effective in treating appendicitis, there is no preventive measure being used at this point. By reengineering Bacteroides to respond to the inflammation of the appendix with a linked de-inflammation, prevention can be possible.
Appendectomy: An emergency surgical operation, appendectomy is the most common treatment for appendicitis. This treatment utilizes laparoscopic instruments to remove the appendix. An incision in the right abdomen allows for proper removal of the appendix. As mentioned earlier, until a few years ago, it was largely believed that the appendix is vestigial and unnecessary. Researchers have recently found the appendix is helpful as it derives a 'safe house' for harmless bacteria. Thereby, as one in every fifteen people have appendicitis in the United States (with appendectomy being the most common pathway of treatment), over 6% of the American population is missing this 'reboot mechanism' for the digestive system. A similar percent of people are affected throughout the western world, with variations occurring as well. Thereby, although appendectomy is extremely effective in removing the inflamed appendix, an individual undergoing the procedure would lose their 'safe house' of bacteria. This can be deemed as not beneficial in many situations.
Appendectomy with Laparoscopic Devices figure a Figure a illustrates the process of removing an individual's appendix. Using laparoscopic instruments, surgeons are essentially able to place an incision in the abdomen to remove the appendix from the intestine.
Antibiotic Therapy: Extremely controversial, this non-operative treatment has recently shown promise in cases of uncomplicated, acute appendicitis. This antibiotic therapy is impactive as it essentially can de-inflame the appendix after symptoms are shown. There have been two different approaches to antibiotic therapy:
The first approach was published in July of 2014; it was called The Nota Study (Non Operative Treatment for Acute Appendicitis): prospective study on the efficacy and safety of antibiotics (amoxicillin and clavulanic acid) for treating patients with right lower quadrant abdominal pain and long-term follow-up of conservatively treated suspected appendicitis). Essentially, researchers clinically used amoxicillin and clavulanic acid to de-inflame the appendix and avoid operation. The study found that the antibiotics were safe and effective in reducing operation rate, complications, as well as overall costs. Researchers tracked the progression of each patients for two years following the intake of the antibiotic, and they found that the recurrences of abdominal pain for patients was less than 14%. Even during these recurrences, the pain could be efficiently treated with further intake of antibiotics (The Nota Study).
The second approach was published in June of 2015, nearly a year after the first antibiotic therapy attempt. This study, Antibiotic Therapy vs Appendectomy for Treatment of Uncomplicated Acute Appendicitis, was published in The Journal of the American Medical Association. Patients in the clinical trial avoided surgical treatment through antibiotic therapy. They each received intravenous ertapenem, oral levofloxacin, and metronidazole as their antibiotic treatment. To determine the antibiotic as successful and effective, researchers set the criterion as patients being discharged from the hospital without the need for surgery as well as no recurring appendicitis within a follow-up period (1 year). Overall, researchers found that this antibiotic treatment marginally did not meet the criterion they had set at the beginning of the study. Yet, most patients did not require appendectomy, and even those who did need appendectomy did not experience any significant complications.
Looking at both of these approaches to antibiotic treatment, it seems as if the first approach, incorporating amoxicillin and clavulanic acid is more effective. An additional study to compare these two approaches against one another could help affirm this, but determining that the first approach seems to be more efficient is plausible.
Figure b illustrates the antibiotic therapy used in the second approach.
Originally, I was hoping to be able to use amoxicillin and clavulanic acid in my APD. Yet, because neither of these are naturally produced as proteins in the human body, it would not be possible to genetically modify a particular gene to secrete both amoxicillin and clavulanic acid when inflammation occurs. With more research, I was able to find that appendicitis is accompanied with an increase in neutrophils, white-blood cells which respond to infection. These neutrophils add to the inflammation, so I realized that inducing apoptosis may help to reduce and remove inflammation. Researchers have found the CD47, a protein entitled Cluster of Differentiation 47, signals neutrophil death (white blood cells). This inspired my design as it became a plausible de-inflammation technique.
Design
There will be two gene sequences that are needed for the APD.
1. C-Reactive Protein Gene: GC01M157474 2.CD47 Gene: GC03M104559
Overview:
A bacteria, Bacteroides, will be reengineered to include the gene encoding for C-Reactive Proteins (CRP) and the gene that encodes for the Cluster of Differentiation 47 (CD47) association protein. Bacteroides will be used because it is in 29% abundance in the appendix.
There will be two genetic pathways in this system. The crux of the first genetic pathway is the production of the CRP. This protein is initially produced in the liver, but generally used as an indicator of inflammation in the heart. The gene encoding for the expression of the protein is, GC01M157474, which will inserted into Bacteroides- directed to the appendix.
The signal molecule for the receptor, in order to produce CRP, will bind in the presence of inflammation in the appendix. After transcription and translation of the gene noted above, CRP will be produced. This protein will act as a regulator for the autoinducer in CD47 production.
The second genetic pathway focuses on the production of CD47 in order to induce neutrophil apoptosis in the appendix. For this to occur, the gene encoded, GC03M104559, will be inserted. Although CD47 already exists in the appendix, this genetic pathway will rely on the existence of CRP to initiate CD47 production.
CRP, produced in the first novel genetic pathway, will bind to a cell-surface receptor so that the gene pathway for CD47 will transcribe and further induce apoptosis of the neutrophils. This, overall, will de-inflame the appendix.
As this process will occur whenever the appendix faces inflammation, de-inflammation will consequently occur- causing a loop between the two stages, and avoiding appendicitis from fully occurring.
Each of the diagrams below depict the process of first creating the C-Reactive Proteins, and then creating the CD47 proteins that will bind to neutrophils in the appendix and cause apoptosis. These genetic occurrences are in the Bacteroides bacteria.
figure cThis diagram portrays the first genetic pathway that will be used to produce the C-Reactive Proteins. In this depiction of the cellular reconstruction within Bacteroides, it is notable that gene GC01M157474 has been added. This gene codes for C-Reactive Proteins when inflammation is prevalent within the structure.
figure dAs shown in this next step, when chemicals are released from the on-start of inflammation, the cell-surface receptor initiates intracellular signaling. At this time, an inducer signal molecule will bind to the activator and allow for the RNA polymerase to bind to the promoter. Transcription and translation will occur and the end product is C-Reactive Proteins which express for inflammation within the appendix.
figure eIn this step, the C-Reactive Proteins were recently produced and they bind to a cell-surface receptor in order to initiate the genetic pathway for CD47 production (gene GC03M104559).
figure fNow that the C-Reactive Proteins bind to the cell-surface receptor, the signal molecule and RNA polymerase bind to the activator and promoter, respectively. Transcription and translation of the gene inserted in Bacteroides allows for CD47 proteins to be produced. These association proteins will interact with the high number of neutrophils in the appendix, during inflammation, in order to induce apoptosis.
figure gNow that the CD47 proteins have been produced, they will bind with a region on the neutrophils in the appendix. As mentioned earlier, the number of neutrophils within the appendix drastically increases during times of inflammation. Past research has shown that these particular association protein can induce apoptosis within neutrophils (white blood cells), while also inhibiting cellular activation. Because these CD47 proteins will both inhibit cellular activation in certain neutrophils, and induce apoptosis in others- inflammation should decrease within the appendix.
figure hFigure h portrays a larger version of the inflamed appendix for better reference and view. After the inflammation first occurs, chemicals will bind to a cell-surface receptor, as shown in figure d, and allow for genetic transcription and translation to produce C-Reactive Proteins. These newly created proteins will bind to another cell-surface receptor to allow for the production of Cluster of Differentiation 47 association proteins, as shown clearly by figure f. Further, as portrayed by figure g, the association proteins will bind to neutrophils in order to inhibit activation and induce apoptosis.
Expected ResultsIf the design works perfectly, then it is expected for C-Reactive Proteins to be produced at the onset of inflammation. After the C-Reactive Proteins are apparent, it would be expected for Cluster of Differentiation 47 to be produced once the inducing signal molecule binds to the activator for the genetic pathway. The presence of this CD47 will inhibit neutrophil activation and eventually cause apoptosis. This apoptosis and reduction of neutrophils in the appendix will result in de-inflammation. These results will bring a successful solution to the problem because it will dedicate a loop of inflammation and de-inflammation. Thereby, appendicitis will be an eliminated ailment... Truth Table
Presence of Inflammation
Presence of CRP
(C-Reactive Proteins)
Presence of CD47
(Cluster of Differentiation 47)
Neutrophil inhibition and apoptosis
De-inflammation
0
0
0
0
0
1
1
1
1
1
Advantages
This biological design is an advantage over the others already out there because there is no design that acts as a prevention mechanism. Moreover, the other 'solutions' to inflammation in the appendix are either appendectomy or antibiotic therapy. The former removes an organ that is a 'safe house' for harmless bacteria, and the latter requires tremendous pain before the antibiotics are effective. Thereby, this solution truly is better than existing technologies- in general. The APD is worth funding because it is the only medically applicable prevention device, and it is also less painful and troublesome than appendectomy as well as antibiotic therapy. Of course a lot of clinical research and funding would be necessary to develop the APD successfully, but it is promising because it is cost effective and requires much less medical intervention at the onset of intervention in the appendix.
There are certainly potential problems in this design, which will have to addressed in order for the APD to be commonly used.
First of all, while the design had been outlined, the procedure for inserting the bacteria would have to be developed much farther. Whether this bacteria would be added in vivo or at the particular age, would have to be designated.
Moreover, Bacteroides survives in the appendix, but it is unknown if it will survive with the two additional genes. As the bacteria's genotype is being altered, ensuring that the redesigned Bacteroides can survive in the appendix is very important to the design.
Another potential problem with this design is that the CD47 binding to the neutrophils could potentially destruct too many neutrophils. These neutrophils are necessary (immune system), but in excess they contribute to unnecessary inflammation.
How would you ensure safety?
To protect employees during the development of this design, it would be beneficial to enforce lab safety. Hazards are largely tangible in this procedure, even though most of the chemicals and proteins being used are not extremely dangerous. If regular safety precaution is taken, the employees working on the development of this design should be alright.
How would you protect the environment during the development of your design?
The environment would not be largely impacted by this design. The synthesized bacteria will be formed in a laboratory setting, and proper regulation will be followed in the controlled area to ensure that the environment is not negatively impacted by the APD.
In what ways could the evolution of cells you've engineered negatively affect use of your design in the future?
Evolution of the cells I've engineered could negatively affect the use of the APD in the future if mutations occur. As the CRP gene is not naturally found in the appendix, it is possible that it does not react as expected. Further, although the CD47 gene is already prevalent in the appendix, its signal molecule for the activator tends to not depend on the presence of a CRP binding a cell-surface receptor. Both of these alterations within Bacteroides could result in problems occurring in the future.
Are there any inefficiencies or shortcomings as proposed by existing technologies?
As mentioned previously, there is no existing technology that allows for appendicitis to be essentially prevented. When the appendix first inflames, it will generate the initiation of the genetic pathways which lead to de-inflammation. Thereby, the appendix will truly not become enlarged enough for a patient to have appendicitis.
As compared to appendectomy, the APD does not remove the appendix. Earlier, it was explained that researchers truly believe that the appendix is important as a safe house for harmless bacteria. On the other hand, several groups of people still believe that it is vestigial. For those, removing the appendix at the onset of inflammation may seem helpful.
There are no shortcomings to the antibiotic therapy.
Does your design pose dangers to the environment, lab safety, and the security of the public?
The design does not pose tangible risks to the environment.
The design does not pose tangible risks to lab safety.
Because of scrutinizing lab testing and regulation, the APD should not and would not pose danger to the security of the general public.
In what ways are the potential rewards worth the risks?
There will always be some risk associated with scientific progress, but the potential rewards that the APD offers truly do outweigh any plausible risks. First off, the APD would prevent appendicitis. In the USA alone, 200,000 people undergo appendectomy each year. With the APD, the appendectomy will no longer need to be a common practice. In fact, rather than millions of people worldwide facing the pain and suffering that comes along with appendicitis, their body would be able to essentially de-inflame without causing as much pain or trauma as that of current practices. Moreover, both the treatments for appendicitis today- appendectomy and antibiotic therapy- are costly. Once the APD is synthesized, its cost will be very minimal and it will be much more effective in totality. Not only can this device prevent full appendicitis from occurring, but it is also cheaper and more sustainable in the long run than current solutions. Thereby, the potential rewards of the APD outweigh plausible risks.
Testing
To test the effectiveness of the system, it would be necessary to first construct a prototype with the CRP gene and the CRP receptor which initiates CD47 production. Then, simulate what occurs in the appendix during the onset of inflammation in a petri dish by inserting inflammation chemicals. Along with the constructed genetic pathways, the existence of chemicals from inflammation in the petri dish should initiate the binding of an inducing signal molecule to an activator in order to transcribe, translate, and synthesize proteins. At the end of the process, the CD47 association proteins should bind to the neutrophils and either inhibit activation or induce apoptosis, if not both processes consecutively. De-inflammation through reduction of the number of neutrophils in the appendix signals a working and an effective device.
Testing would help to improve the system as it would show if there are particular gaps that prevent the system from properly functioning. Moreover, it would help reveal greater potential for the system as variations in results could find if altering more or less of the bacteria's genotype may positively help the APD, for example. Another possibility for greater potential could root from an unexpected result at the end of the procedure, one which may be helpful to the APD's overall design.
Works Cited
"Appendicitis Symptoms, Causes, Surgery, and Recovery." WebMD. WebMD. Web. 04 Aug. 2016.
"Appendicitis Therapy." Womens Best. Web. 04 Aug. 2016.
"Appendicitis." Wikipedia. Wikimedia Foundation. Web. 04 Aug. 2016.
"Cluster of Differentiation 47 Gene." Gene Cards. Web. 04 Aug. 2016.
"C-reactive Protein." C-reactive Protein: MedlinePlus Medical Encyclopedia. Web. 04 Aug. 2016.
"C-Reactive Protein Gene." Gene Cards. Web. 04 Aug. 2016.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.
Leigh, D. A., K. Simmons, and E. Norman. "Bacterial Flora of the Appendix Fossa in Appendicitis and Postoperative Wound Infection." Journal of Clinical Pathology 27.12 (1974): 997-1000. Web.
"Mechanisms of CD47-induced Caspase-independent Cell Death in Normal and Leukemic Cells: Link between Phosphatidylserine Exposure and Cytoskeleton Organization." Home. Web. 03 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 02 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 03 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 03 Aug. 2016. National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.
My name is Ananya Kachru, and I am a rising junior at Amity High School in CT. I decided to participate in the Biological Research program at the Boston Leadership Institute because I am passionate about science research, and because I love biology! My love for biology started when I took a biology class my freshman year at AHS. It quickly became my absolute favorite- I loved it all. Biology intrigues me because it literally is life. It is the science behind every single living thing… it truly amazes me. I am looking forward to taking AP Biology next year! Moreover, ever since the 8th grade, I have been involved with science research. In middle school, I worked on social robotics and created a writing robot called WriteBot (I know, I should have come up with a more creative name!) In my freshman and sophomore years, I worked a lot on behavioral research with cognitive neuroscience. For my junior year, I am looking to expand my horizons and finally do biological research! Science research is truly the crux of modern society, and I believe that the constant revelations being made are truly incredible.
In school, I am involved in several clubs and organizations. I am really, really passionate about debate, and I have been a part of the debate team at my high school since freshman year. I love debate because although it is extremely competitive, it fosters a collaborative environment. Earlier this summer, I was at debate camp. My debate partner and I were fortunate enough to break into quarterfinals at the Calvin Coolidge National Debate Tournament, and all our camp friends helped us intensely prepare for quarterfinals- which I believe really exemplifies the collaborative atmosphere. Also, last year, I founded my school’s Academic Decathlon team along with one of my classmates. Starting the team from ground up and persevering along with 7 amazing teammates to the E-National competition was an incredibly unique experience. ALSO- the science topic for AcDec last year was biology!!! Student Government, Infinite Possibilities (a STEM mentoring program), and Kids v. Cancer are a few of the organizations I am also very involved with at my school. Outside of school, I enjoy volunteering as an assistant dance teacher at my dance school, as a research intern at the Haskins Research Laboratories, and as a teen teacher at a local elementary school (little kids are the best). I also am passionate about youth activism, Kathak dance (Indian classical dancing), and pageantry.
Some fun facts about me:
Favorite food: That is really hard- but probably Margherita pizza from Frank Pepe’s (very famous in CT) coupled with a really good Caesar salad.
Favorite dessert: CHEESECAKE
Favorite TV show: I LOVE Gilmore Girls. But, I have also been a loyal Dance Moms viewer since Season 1. (If any of you are DM fans- I have gone back and forth over Team Chloe and Team Maddie for years… and them both being nominated for TCA Choice Dancer for the second year in a row is so excited ah)!
Favorite movie: The Sisterhood of the Traveling Pants
Favorite color: Hot pink
Favorite Harry Potter house: Gryffindor, of course!
Favorite board game: Either LIFE or RISK
Favorite book: Although I first read it in English Class, I absolutely love The Great Gatsby. I also love the Mother-Daughter Book Club books. I grew up reading them, and they still remain to be some of my favorite books.
Favorite season: Winter! I am a December baby, and literally all of my family’s major occasions happen in the last three months of the year. I also really love New England winters!
Favorite musical: HAMILTON (I am counting down days until I see it!)
I look forward to meeting you all soon!
Ananya Kachru
BLI Session II
Biological Research
Appendicitis Prevention Device (APD)
Purpose
Problem: Appendicitis is the inflammation of the appendix through pus, followed with much pain. It tends to occur when the appendix is blocked- by stool, an infection, foreign bacteria presence, or even cancer. Interestingly, the appendix is often considered to be vestigial and unimportant, but recent studies have shown that there is a particular significance to the appendix. Currently, researchers have found that the appendix essentially acts as a ‘safe house’ for unharmful bacteria. For example, when an individual suffers from dysentery or cholera, the appendix can activate as a ‘reboot mechanism’ for the digestive system. As one in every fifteen people suffer from appendicitis, it is important to design a preventive measure to preserve the appendix.
Importance: It is crucial to solve this problem because as there is currently no functioning preventive measure for appendicitis, so anyone who suffers from the illness must undergo appendectomy or receive antibiotic therapy. Appendectomy is a common procedure, but complications are evidently plausible and it is a significant inconvenience. On the other hand, antibiotic therapy is once again not very effective and causes much pain within individual patients. Providing a preventive measure for the general population will give ease and more comfort. Moreover, determining potential devices in the health field often comes down to cost-effectiveness and quality. If a preventive mechanism is synthetically designed, appendectomy and antibiotic therapy will no longer have to be as prevalent in the health industry and a lot of financial burden will be lifted from potential patients.
Address Problem: The design that I am creating will essentially prevent appendicitis by allowing for a loop between inflammation and de-inflammation. By reengineering Bacteroides, the most abundant bacteria in the appendix, the APD design will largely focus on the prevalence of C-Reactive Proteins (CRPs). These proteins generate in the liver, but are most typically recognized as a marker of inflammation in the cardiovascular system. The APD will utilize the gene that encodes for this protein, in order to express CRPs at the site of inflammation in the appendix rather than the heart. Further, a signal molecule will only bind to a particular receptor for the production of Cluster of Differentiation 47 proteins (CD47) when the CRPs are prevalent in the genetic pathway. CD47 leads to de-inflammation of the appendix. Thereby, whenever inflammation is present, CRPs are produced. When CRPs are produced, CD47 for de-inflammation is synthesized. This design ensures that whenever the appendix is inflamed, it can and will be de-inflamed. With this regular cycle between the two stages, appendicitis can be prevented.
Competing Technologies
Currently, appendicitis is dealt with through two methods- primarily an appendectomy procedure, but recently even an antibiotic therapy (especially with patients enduring uncomplicated acute appendicitis). While both of these methods are effective in treating appendicitis, there is no preventive measure being used at this point. By reengineering Bacteroides to respond to the inflammation of the appendix with a linked de-inflammation, prevention can be possible.Appendectomy: An emergency surgical operation, appendectomy is the most common treatment for appendicitis. This treatment utilizes laparoscopic instruments to remove the appendix. An incision in the right abdomen allows for proper removal of the appendix. As mentioned earlier, until a few years ago, it was largely believed that the appendix is vestigial and unnecessary. Researchers have recently found the appendix is helpful as it derives a 'safe house' for harmless bacteria. Thereby, as one in every fifteen people have appendicitis in the United States (with appendectomy being the most common pathway of treatment), over 6% of the American population is missing this 'reboot mechanism' for the digestive system. A similar percent of people are affected throughout the western world, with variations occurring as well. Thereby, although appendectomy is extremely effective in removing the inflamed appendix, an individual undergoing the procedure would lose their 'safe house' of bacteria. This can be deemed as not beneficial in many situations.
figure a
Figure a illustrates the process of removing an individual's appendix. Using laparoscopic instruments, surgeons are essentially able to place an incision in the abdomen to remove the appendix from the intestine.
Antibiotic Therapy:
Extremely controversial, this non-operative treatment has recently shown promise in cases of uncomplicated, acute appendicitis. This antibiotic therapy is impactive as it essentially can de-inflame the appendix after symptoms are shown.
There have been two different approaches to antibiotic therapy:
- The first approach was published in July of 2014; it was called The Nota Study (Non Operative Treatment for Acute Appendicitis): prospective study on the efficacy and safety of antibiotics (amoxicillin and clavulanic acid) for treating patients with right lower quadrant abdominal pain and long-term follow-up of conservatively treated suspected appendicitis). Essentially, researchers clinically used amoxicillin and clavulanic acid to de-inflame the appendix and avoid operation. The study found that the antibiotics were safe and effective in reducing operation rate, complications, as well as overall costs. Researchers tracked the progression of each patients for two years following the intake of the antibiotic, and they found that the recurrences of abdominal pain for patients was less than 14%. Even during these recurrences, the pain could be efficiently treated with further intake of antibiotics (The Nota Study).
- The second approach was published in June of 2015, nearly a year after the first antibiotic therapy attempt. This study, Antibiotic Therapy vs Appendectomy for Treatment of Uncomplicated Acute Appendicitis, was published in The Journal of the American Medical Association. Patients in the clinical trial avoided surgical treatment through antibiotic therapy. They each received intravenous ertapenem, oral levofloxacin, and metronidazole as their antibiotic treatment. To determine the antibiotic as successful and effective, researchers set the criterion as patients being discharged from the hospital without the need for surgery as well as no recurring appendicitis within a follow-up period (1 year). Overall, researchers found that this antibiotic treatment marginally did not meet the criterion they had set at the beginning of the study. Yet, most patients did not require appendectomy, and even those who did need appendectomy did not experience any significant complications.
Looking at both of these approaches to antibiotic treatment, it seems as if the first approach, incorporating amoxicillin and clavulanic acid is more effective. An additional study to compare these two approaches against one another could help affirm this, but determining that the first approach seems to be more efficient is plausible.figure b
Figure b illustrates the antibiotic therapy used in the second approach.
Originally, I was hoping to be able to use amoxicillin and clavulanic acid in my APD. Yet, because neither of these are naturally produced as proteins in the human body, it would not be possible to genetically modify a particular gene to secrete both amoxicillin and clavulanic acid when inflammation occurs. With more research, I was able to find that appendicitis is accompanied with an increase in neutrophils, white-blood cells which respond to infection. These neutrophils add to the inflammation, so I realized that inducing apoptosis may help to reduce and remove inflammation. Researchers have found the CD47, a protein entitled Cluster of Differentiation 47, signals neutrophil death (white blood cells). This inspired my design as it became a plausible de-inflammation technique.
Design
There will be two gene sequences that are needed for the APD.1. C-Reactive Protein Gene: GC01M157474
2.CD47 Gene: GC03M104559
Overview:
- A bacteria, Bacteroides, will be reengineered to include the gene encoding for C-Reactive Proteins (CRP) and the gene that encodes for the Cluster of Differentiation 47 (CD47) association protein. Bacteroides will be used because it is in 29% abundance in the appendix.
- There will be two genetic pathways in this system. The crux of the first genetic pathway is the production of the CRP. This protein is initially produced in the liver, but generally used as an indicator of inflammation in the heart. The gene encoding for the expression of the protein is, GC01M157474, which will inserted into Bacteroides- directed to the appendix.
- The signal molecule for the receptor, in order to produce CRP, will bind in the presence of inflammation in the appendix. After transcription and translation of the gene noted above, CRP will be produced. This protein will act as a regulator for the autoinducer in CD47 production.
- The second genetic pathway focuses on the production of CD47 in order to induce neutrophil apoptosis in the appendix. For this to occur, the gene encoded, GC03M104559, will be inserted. Although CD47 already exists in the appendix, this genetic pathway will rely on the existence of CRP to initiate CD47 production.
- CRP, produced in the first novel genetic pathway, will bind to a cell-surface receptor so that the gene pathway for CD47 will transcribe and further induce apoptosis of the neutrophils. This, overall, will de-inflame the appendix.
As this process will occur whenever the appendix faces inflammation, de-inflammation will consequently occur- causing a loop between the two stages, and avoiding appendicitis from fully occurring.Each of the diagrams below depict the process of first creating the C-Reactive Proteins, and then creating the CD47 proteins that will bind to neutrophils in the appendix and cause apoptosis. These genetic occurrences are in the Bacteroides bacteria.
figure cThis diagram portrays the first genetic pathway that will be used to produce the C-Reactive Proteins. In this depiction of the cellular reconstruction within Bacteroides, it is notable that gene GC01M157474 has been added. This gene codes for C-Reactive Proteins when inflammation is prevalent within the structure.
figure dAs shown in this next step, when chemicals are released from the on-start of inflammation, the cell-surface receptor initiates intracellular signaling. At this time, an inducer signal molecule will bind to the activator and allow for the RNA polymerase to bind to the promoter. Transcription and translation will occur and the end product is C-Reactive Proteins which express for inflammation within the appendix.
figure eIn this step, the C-Reactive Proteins were recently produced and they bind to a cell-surface receptor in order to initiate the genetic pathway for CD47 production (gene GC03M104559).
figure fNow that the C-Reactive Proteins bind to the cell-surface receptor, the signal molecule and RNA polymerase bind to the activator and promoter, respectively. Transcription and translation of the gene inserted in Bacteroides allows for CD47 proteins to be produced. These association proteins will interact with the high number of neutrophils in the appendix, during inflammation, in order to induce apoptosis.
figure gNow that the CD47 proteins have been produced, they will bind with a region on the neutrophils in the appendix. As mentioned earlier, the number of neutrophils within the appendix drastically increases during times of inflammation. Past research has shown that these particular association protein can induce apoptosis within neutrophils (white blood cells), while also inhibiting cellular activation. Because these CD47 proteins will both inhibit cellular activation in certain neutrophils, and induce apoptosis in others- inflammation should decrease within the appendix.
figure hFigure h portrays a larger version of the inflamed appendix for better reference and view. After the inflammation first occurs, chemicals will bind to a cell-surface receptor, as shown in figure d, and allow for genetic transcription and translation to produce C-Reactive Proteins. These newly created proteins will bind to another cell-surface receptor to allow for the production of Cluster of Differentiation 47 association proteins, as shown clearly by figure f. Further, as portrayed by figure g, the association proteins will bind to neutrophils in order to inhibit activation and induce apoptosis.
Expected ResultsIf the design works perfectly, then it is expected for C-Reactive Proteins to be produced at the onset of inflammation. After the C-Reactive Proteins are apparent, it would be expected for Cluster of Differentiation 47 to be produced once the inducing signal molecule binds to the activator for the genetic pathway. The presence of this CD47 will inhibit neutrophil activation and eventually cause apoptosis. This apoptosis and reduction of neutrophils in the appendix will result in de-inflammation.
These results will bring a successful solution to the problem because it will dedicate a loop of inflammation and de-inflammation. Thereby, appendicitis will be an eliminated ailment...
Truth Table
(C-Reactive Proteins)
(Cluster of Differentiation 47)
Advantages
This biological design is an advantage over the others already out there because there is no design that acts as a prevention mechanism. Moreover, the other 'solutions' to inflammation in the appendix are either appendectomy or antibiotic therapy. The former removes an organ that is a 'safe house' for harmless bacteria, and the latter requires tremendous pain before the antibiotics are effective. Thereby, this solution truly is better than existing technologies- in general.The APD is worth funding because it is the only medically applicable prevention device, and it is also less painful and troublesome than appendectomy as well as antibiotic therapy. Of course a lot of clinical research and funding would be necessary to develop the APD successfully, but it is promising because it is cost effective and requires much less medical intervention at the onset of intervention in the appendix.
Potential Problems (and Frequently Asked Questions)
There are certainly potential problems in this design, which will have to addressed in order for the APD to be commonly used.How would you ensure safety?
How would you protect the environment during the development of your design?
In what ways could the evolution of cells you've engineered negatively affect use of your design in the future?
Are there any inefficiencies or shortcomings as proposed by existing technologies?
Does your design pose dangers to the environment, lab safety, and the security of the public?
In what ways are the potential rewards worth the risks?
Testing
To test the effectiveness of the system, it would be necessary to first construct a prototype with the CRP gene and the CRP receptor which initiates CD47 production. Then, simulate what occurs in the appendix during the onset of inflammation in a petri dish by inserting inflammation chemicals. Along with the constructed genetic pathways, the existence of chemicals from inflammation in the petri dish should initiate the binding of an inducing signal molecule to an activator in order to transcribe, translate, and synthesize proteins. At the end of the process, the CD47 association proteins should bind to the neutrophils and either inhibit activation or induce apoptosis, if not both processes consecutively. De-inflammation through reduction of the number of neutrophils in the appendix signals a working and an effective device.Testing would help to improve the system as it would show if there are particular gaps that prevent the system from properly functioning. Moreover, it would help reveal greater potential for the system as variations in results could find if altering more or less of the bacteria's genotype may positively help the APD, for example. Another possibility for greater potential could root from an unexpected result at the end of the procedure, one which may be helpful to the APD's overall design.
Works Cited
"Appendicitis Symptoms, Causes, Surgery, and Recovery." WebMD. WebMD. Web. 04 Aug. 2016.
"Appendicitis Therapy." Womens Best. Web. 04 Aug. 2016.
"Appendicitis." Wikipedia. Wikimedia Foundation. Web. 04 Aug. 2016.
"Cluster of Differentiation 47 Gene." Gene Cards. Web. 04 Aug. 2016.
"C-reactive Protein." C-reactive Protein: MedlinePlus Medical Encyclopedia. Web. 04 Aug. 2016.
"C-Reactive Protein Gene." Gene Cards. Web. 04 Aug. 2016.
"Early Appendicitis Symptoms, Signs, Pain, Test, & Surgery." MedicineNet. Web. 04 Aug. 2016.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.
"Genes and Mapped Phenotypes." National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.
Leigh, D. A., K. Simmons, and E. Norman. "Bacterial Flora of the Appendix Fossa in Appendicitis and Postoperative Wound Infection." Journal of Clinical Pathology 27.12 (1974): 997-1000. Web.
"Mechanisms of CD47-induced Caspase-independent Cell Death in Normal and Leukemic Cells: Link between Phosphatidylserine Exposure and Cytoskeleton Organization." Home. Web. 03 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 02 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 03 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 03 Aug. 2016.
National Center for Biotechnology Information. U.S. National Library of Medicine. Web. 04 Aug. 2016.